Design, fabrication, and decontamination of low-cost microfluidics for nucleic acid amplification

• Innovative reaction chamber design to reduce bubble formation when filling • Low-cost fabrication and decontamination methods for microfluidic diagnostic devices • Guidelines for material selection and compatibility with nucleic acid amplification Liquid assays, such as polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP), are difficult to implement in point-of-care microfluidic devices because bubbles often form. We present a reaction chamber design called same-depth inlet outlet (SDIO), which was found to reduce the instances of bubble formation by 92.2% across different flow rates when compared to traditional designs. We developed with low resource settings in mind, so our design uses xurography and laser cutting techniques, which keep costs low and are easier for unskilled personnel to assemble through lamination. The fabrication methods were tested for nuclease contamination and best practices to reduce nucleases during and after chip assembly are detailed. Our technique using a combination of ethanol rinses and ultraviolet-C light radiation was found to reduce RNases up to tenfold. Different materials were tested for microfluidic compatibility with LAMP assay reagents by making chips that realistically emulate final surface areas and volumes. A variation in performance was found among different adhesives, where the best fluorescence ratio between positive and negative reactions was 4.63. Our microfluidic design was validated by amplifying a spiked RT-LAMP assay with SARS-CoV-2 primers in the reaction chambers. The overall findings are geared toward facilitating early-stage development and producing prototypes with consistent, reliable results.